The long-term goal of this research is to determine the molecular and cellular mechanisms responsible for the regulation of muscarinic acetylcholine receptor (mAChR) gene expression in the retina during development. Muscarinic receptors have an important role in the processing of visual information in the retina, and have also been implicated in the development of the embryonic retina. Our laboratory has found that there is receptor subtype- and cell-specific regulation of mAChR in the chick retina during embryonic development. The developmental regulation of the M2 receptor-in vivo can be mimicked in retina cell culture by an apparently novel developmentally-regulated secreted factor. This application will identify and determine the mechanism of action of the M2-inducing factor. The specific aims are: (1) To purify the M2 receptor-inducing factor. We have developed a serum-free culture system that supports secretion of this factor, and have used a rapid reporter gene assay to obtain highly enriched preparations of this factor. We will refine this purification scheme to purify the factor to sufficient homogeneity to allow biochemical and molecular biological analyses. (2) To isolate cDNA clones encoding the M2 receptor-inducing factor. We will use both a protein sequence determination approach and expression cloning to isolate cDNA clones encoding the factor. (3) To test the hypothesis that the M2 receptor-inducing factor regulates M2 receptor expression in vivo. We will determine if the factor identified on the basis of its ability to induce M2 receptor expression in retinal cells in culture is indeed responsible for the developmental induction of retinal M2 receptor expression in vivo. (4) To determine the molecular and cellular mechanisms responsible for the regulation of the M2 receptor by the M2 receptor-inducing factor. These experiments should provide valuable new information on the mechanisms responsible for the developmental regulation of neurotransmitter receptor gene expression in the retina.